This invention relates to the isolation and purification of nucleic acids from cells, and particularly to apparatus for automatically achieving such isolation.
One of the first steps in the in vitro manipulation of nucleic acids involves their isolation. For example, relatively pure samples of genomic DNA are required in order to perform tests for genetic desease and recombinant technology requires isolation of both the vector DNA and the DNA to be cloned.
As a general rule, DNA does not exist as a free molecule in a cell, but instead exists as a complex association of DNA, RNA and proteins. This is a consequence of the role of DNA as the genetic information, the DNA is used as a template for the production of messenger RNA, which is translated by the ribosome into protein. Proteins directly involved in the process of gene expression, such as RNA polymerase and regulatory proteins, interact with DNA in vivo to form nucleo-protein complexes, DNA polymerase, DNA ligase, various unwinding and supercoiling enzymes, recombination and repair enzymes, and those proteins involved in the initiation or maintenance of DNA replication are also associated with DNA in vivo and hence complicate the isolation of pure DNA. Because of this complex association of DNA with these other proteins and nucleic acids, the purification (isolation) approach for obtaining DNA can generally be thought of as a three step process: (1) releasing soluble, high molecular weight DNA from disrupted cell wall and membranes; (2) dissociating DNA-protein complexes by protein denaturation or proteolysis; and (3) separating DNA from the other macromolecules.
Within this process, DNA of bacterial origin (prokaryotic DNA) is typically purified by different methods, depending on whether the DNA is chromosomal DNA, the bacterial cell wall is generally weakened by freeze-thawing or by treatment with the enzyme lysozyme and the chelating agent ethylenediaminetetraacetic acid (EDTA). Cell lysis is accomplished by the addition of a detergent such as sodium dodecyl sulfate (SDS) in a buffered saline solution. Following lysis, the solution is treated with pancreatic ribonuclease to hydrolyze RNA and protease to degrade proteins. Residual proteins and oligopeptides are extracted with an organic solvent, such as phenol or an equal mixture of phenol and chloroform. Most of the protein will denature and enter the organic phase or precipitate at the interface of the organic and aqueous phases, this phase separation being accomplished by means of centrifugation. The clear, viscous aqueous phase containing the DNA is then removed. With the addition of alcohol, the DNA precipitates out of the aqueous phase as a white fibrous material and can be spooled onto a glass rod. Precipitation from alcohol serves to concentrate the high molecular weight DNA while removing the small oligonucleotides of DNA and RNA, detergent, and the organic solvent used in the removal of proteins. Residual detergent and salts can be removed by dialysis of the resuspended DNA solution against the desired buffer. In some instances, it may be desirable to further purify the DNA by centrifugation on isopycnic cesium chloride gradients, or by hydroxylapatite chromatography. In the above process for chromosomal DNA, typical protocols often require at least two days for the DNA extraction and purification process. (See Recombinant Techniques by Raymond L. Rodrigues, and Robert C. TAct, 1983, p. 162).
During the purification of extrachromosomal elements of prokaryotic DNA, including plasmids and bacteriophage, it is desirable to minimize the amount of chromosomal DNA contaminating the preparation. With Bacteriophage, this is often accomplished by first purifying the phage particles from the infected bacteria, then treating the purified phage particles with protease and/or phenol to release the bacteriophage DNA. Further purification of the DNA is accomplished by means similar to those described for chromosomal DNA. Due to its size, however, precipitated bacteriophage and plasmid DNA cannot be spooled out on a glass rod and is therefore generally recovered by centrifugation. Again, three days is not atypical for the entire isolation and purification process.
For eukaryotic cells, isolation and purification of total cellular DNA is often achieved by a modification of the detergent lysis procedure described above for bacteria. The key difference is that typically cell lysis and digestion of cellular proteins are accomplished using proteinase K in the presence of the detergent. (See M. Gross-Bellard, P. Oudet, and P. Chambon, Eur. J. Biochem., 36 (1973) 32-38; N. Blin, and D. W. Stafford, Nuc. Acid. Res., 3 (1976) 2303-2308; and D. J. Law, P. M. Frossard and D. L. Ruchnagel, Gene, 28 (1984) 153-158. The proteinase K is then removed by extraction of the lysate with phenol or a phenol/chloroform mixture. Typically, in the mixing process as for the extraction of bacterial DNA, the lysate/phenol or lysate/phenol-chloroform forms an emulsion, the aqueous and organic phases of which are separated by centrifugation. The upper, or aqueous, phase containing the DNA is then poured off or removed using a pipette, and this essentially protein-free lysate is dialyzed to remove small molecular weight cellular contaminants and residual phenol.
In the above approaches, a major limitation on the extraction which critically limits the ability to automate the process, is the need for centrifugation to separate the aqueous and organic phases during the phenol extraction. Often several extractions are required to achieve the desired purity, each one requiring centrifugation. Largely due to these various centrifugations, the work is performed manually and is therefore expensive. Also these configurations make automating of the extraction process difficult and expensive.